Dihydrodaunomycin antibiotic and derivatives thereof

ABSTRACT

DESCRIBED IS A MICROBIOLOGICAL PROCESS FOR THE PREPARATION OF THE ANITBIOTIC SUBSTANCES DIHYDRODAUNOMYCIN, DAUNSOMAINILDAUNOMYCIN AND DIHYDRODAUNOMUCINONE AND THEIR SALTS WITH PHARMACEUTICALLY ACCEPTABLE NON-TOXIC ACIDS. THE PROCESS COMPRISES GROWING THE MICROOGANISM STREPTOMYCES PEUCETIUS VAR. CARNES UNDER AEROBIC CONDITIONS.

United States Patent @fice 3223, 115:

MICROSCOPIC CHARACTERISTICS 3,686,163

Th v t' mn o wr z ggggggg mi. ii iiill 131i???kiig fifiiiikfi i 6 3 Federico Arcarnone, Milan, Giuseppe Cassinelli, Rivanazi and branched .These ralmlficatlons bear t.h1.cker mm, and Sergio Pence and Luigi Tognoli, Milan, Italy 5 p ae (1.1-1.6 m thick), which are called COIIIdIOPhO IB S. assignors to Societa Farmaceutici Italia, Milan, Italy These are Often Spherical and elidilig 111 hooks- The Conldla No Drawing, Filed May 13, 1969, S N 824,282 have a sphencal form and a diameter of from 1.8 to 3.3 Claims priority, applicatior/lfiltaly, May 14, 1968, mg. They are first disposed in little chains, then free. 6 444 8 Under the electron microsco e, the conidia show a form Int. Cl. C07c 47/18 10 nearly spherical with irregular surroundings and warty U.S. Cl. 260-210 AB 6 Claims surfam MACROSCOPIC CHARACTERISTICS Table 1 reports the macroscopic characteristics, on the indicated media, by growing the microorganism at 28 C. and carrying out observations 3, 8, 15, 21 and 30 days after inoculation.

ABSTRACT OF THE DISCLOSURE 5 Described is a microbiological process for the preparation of the antibiotic substances dihydrodaunomycin, daunosaminildaunomycin and dihydrodaunomycinone and BIOCHEMICA L CHARACTERISTICS their salts with pharmaceutically acceptable non-toxic Geiailnei total y lf acids. The process comprises growing the microorganism Starch: total hydrolysis Streptomyces peucetius var. carneus under aerobic con- Niifatesl I10 reduction to es ditions. H 8: slight production Melanoid pigments: no production Our invention relates to antibiotic substances, their de- 25 Milk: nelther coagulauon nor peptomzatlon rivatives and the preparation thereof. More particularly, The mlcroorganism utllizesf tyrosme: dxylose maltose the new antibiotic substances of the indicator type called mannose manmtol, glycerm, 9 saccharose dihydrodaunornycin, danuosarninildaunomycin, their salts, alose raffinoei fructose and 'f 1t f not i the aglycone dihydrodaunomycinone and their preparation adomtol ramnose sofbltoli l'arabmose mesom' from cultures of Streptomyces peucetius var. carneus are 031ml and esekllme- In hqu1d W'Q and aerated described. The new antibiotic substances dihydrodaunoculture the Stram Produces antlblotlc substances mycin, daunosaminildaunornycin and dihydrodaunomyci- IDENTIFICATION OF THE STRAIN none, which We have als na d r pecti ly 1, The description of the microorganism under examina- B107 and 13-112, Possess interesting Pharmacological tion places it in the classification system of Pridham et a1. activities particularly as antitumoral, antibacteric, antiviral (Appl, Mi bi l 6, 52, 195 i h Section Retinaand antiprotozoal agents. culum apertum. In the classification system of Baldacci The new strain Streptomyces peucetius var. cameus (Giorn. di MiC P 10, 1 the microorganwhich produces the new antibiotics, called also Streptoism belongs to the series Albosporeus. In the system of TABLE 1. Cultural characteristics of Streptomyces peucetiux var. comma Growth Aerial mycelium Vegetative mycelium Soluble pigments Medium Little confluent colonies Very scarce, smooth, green- Malt yeast extract agar (Sec. with wrinkled folds, hard,, gray colored; absence of Abundant, yellowish, then Intense first red-yellow,

Hesseltine et al.). 1 reheved, abundant. spirals and verticils. red-yellow. then rown-red.

Bonnet agar Scarce with li tle single Absent Fair, vinaceous pink Absent.

yellowish colonies. Emerson agar Fair with lit le confl ent d0 Fair, colorless Reddish-plae brown.

colonies 1n lichenoid, no hard patina. Potato agar (Sec. Hesseltine Abundant in smooth uni- Abundant, blue-green-gray; Abundant, flesh-colored; Intense, first reddish-yellow,

et al.). 1 form patina. hook-ended and then smooth, hard patina. then darkbrange.

v loop-ended hyphae. Peptone agar KNO Abu ndant 1n httle confluent Absent Abundant, colorless Absent.

co omes. Czapek agar Abundant in little confluent scanty, dirty white, Abundant, bright fiesh- Do.

colonies. slightly cottony; hook and pinkcolored.

loop-onded hyphae. Asparagine-glucose agar Scauty in smgle little scanty, whitish-pink; very Scanty, colorless Do.

colonies. broken mycelium, short, without apical hooks. Glycerin-glycine agar Abundant in smooth, hard Absent Abundant from yellow to Do;

patina. light orange, Starch agar scanty in single little col- Fair, from blue-green to scanty, colorless then plnk- Do.

om'es. gray-green. yellowish. Gclatine Moderate in surface Absent Moderate, from colorless to Abundant, dark brownyellowish. black. Milk scanty ..d0 Scanty, ring-formed in Scanty, pink.

surface, rosesahnon colored.

1 Hesseltiue et 2.1., 1954 Ann. N.Y. Acad. Sci. 60, pp. 136-151.

myces F.I.65 (number of the strain collection of Societa Waksman (The Actinomycetes, vol. II, pg. 129, 1961) the Farmaceutici Italia) has been deposited, without reservamicroorganism belongs to the series Ruber. A comparison tion, at the Institute of Plant Pathology of the University between the characteristics of the microorganism and of Milan receiving the number I.P.V. 1950; at the Amerithose of species belonging to the cited systematic groups can Type Culture Collection, Rockville, Md. 20852, re- (Taxa), has shown that none of them has characteristics ceiving the index number A.T.C.C. 21354, and the Comcorresponding to those of the microorganism under exmonwealth Mycological Institute, Ferry Lane, Kew, Suramination. Table 2 reports the data of this comparison as rey, England, receiving the index number I.M.I. 136,533. for the species producing substances similar to those we It shows the following characteristics. have studied. In the table are data referred to Streptomyces myces peucetiuis' var. carneus, in a cultural medium containing carbon, nitrogen sources and mineral salts, some new antibotic substances have been obtained which possess antitumoral, antibacteric, antiviral and antiprotozoal activity. More particularly, the dihydrodaunomycin, daunosaminildaunomycin and dihydrodaunomycinone production is carried out by cultivating Streptomyces p'eucetius var. carneus in a liquid cultural medium, previously sterilized, under aerobic conditions, from 25 to 35 C., over a period of from 4 to 7 days, at a pH of from 6.5 to 8.

The cultural medium consists of a carbon and a nitrogen source and mineral salts. The carbon source may consist of starch, dextrine, glucose, glycerin, mannite, maltose, corn steep liquor, distillers solubles, soybean oil and soybean meal. The nitrogen source may consist, besides the complex substances containing nitrogen mentioned above, of dry yeast, flesh peptone and/or casein. Good results are also obtained using ammonium salts such as nitrate, sulphate and diphosphate. Suitable mineral salts for the production of antibiotics vary according to the medium employed. Calcium carbonate is almost always present and chlorides, sulphates, phosphates, etc. of sodium, potassium, magnesium, manganese, iron, copper, zinc and cobalt can be added.

The fermentation is carried out in Erlenmeyer flasks or in laboratory and industrial fermenters of various capacity. When the fermentation is over, the antibiotic substances dihydrodaunomycin, daunosaminildaunomycin and dihydrodaunomycinone are extracted from the culture broth, with suitable solvents under suitable conditions and separated by chromatography and then purified. The quantity of antibiotics present in the culture broths is determined spectrophotometrically and then compared with samples of known antibiotics.

CHEMICAL-PHYSICAL PROPERTIES The antibiotic substances dihydrodaunomycin, daunosaminildaunomycin and dihydrodaunomycinone show the following chemical structure:

wherein the formula represents the antibiotic dihydrodaunomycin when R is and R is the radical daunosaminil and R is a hydrogen atom.

The elementary analysis of the dihydrodaunomycin hydrochloride gives the following values:

Empirical formula: C H 0 N-HCl.Found (percent): C, 57.16; H, 6.01; N, 2.68; CI, 5.86. Calculated (percent): C, 57.29; H, 5.71; N, 2.47; Cl, 6.26.

The absorption spectrum in the U.V. and in the visible ranges gives, in methanol, adsorption maxima at the following wave lengths:

Important bands are observed in the LR. spectrum in KBr at the following wave lengths (in ,u): 2.94; 3.44; 6.19; 6.32; 6.95; 7.11; 7.25; 7.80; 8.28; 8.95; 9.40; 9.95; 10.18; 11.5; 12.30; 12.65; 13.18.

The elementary analysis of the daunosaminildaunomycin hydrochloride gives the following values:

Empirical formula: C H 0 N -2HCL Found percent: C, 54.42; H, 6.17; N, 3.64; CI, 9.43. Calculated percent: C, 54.32; H, 5.80; N, 3.84; Cl, 9.72.

The adsorption spectrum in the U.V. and in the visible ranges gives, in methanol, adsorption maxima at the following wave lengths:

Bands are observed in the IR. spectrum at the following wave lengths (in m 2.93 (wide); 3.44 (wide); 5.80; 6.18; 6.30; 7.10; 7.78; 8.24; 8.95; 10.05; 12.20; 12.50; 13.10.

Dihydrodaunomycinone may be obtained not only from the culture broths of the Streptomyces peucetius var. carneus, but also by acid hydrolysis of the antibiotic dihydrodaunomycin; its elementary analysis gives the following values:

Empirical formula: C H O Found percent: C, 62.83; H, 5.16; OCH 7.75. Calculated percent: C, 62.90; H, 5.02; 0CH 7.75.

The adsorption spectrum in the U.V. and in the visible ranges gives, in methanol, adsorption maxima at the following wave lengths:

In the LR. spectrum are observed remarkable bands at the following wave lengths (in ,u): 2.94; 3.44; 6.19; 6.35; 6.92; 7.08; 7.30; 7.84; 8.09; 8.31; 9.38; 9.80; 10.20; 11.38; 12.50; 13.18.

ANTITUMORAL ACTIVITY The antitumoral activity has been tested in vitro on explants of Sarcoma MC, Hela cells and fibroblasts. It has been calculated the Inhibiting Dose (ID' which means the concentration of the substance capable of inhibiting the mitotic division in vitro of 50% of the tested cells.

Percent reduction of the tumor development Mammary carcinoma C3H/He mouse 16% (5 mg./kg./die during 50% (5 mgJkg/die/two 8 days subcutaneously). cycles of 6 days each and 5 days interval). 54% (7 mgJkgJdie during 8 days intravenously).

Tested substance Sarcoma 180 mouse Daunosarninildaunomyein.

Dihydrodaunomyein.

ANTIBACTERIAL ACTIVITY The antibacterial activity has been tested in vitro on Bacillus mycoides and the Minimum Inhibiting Dose (MID) which means the lowest quantity of the substance capable of totally inhibiting the development in vitro of the tested organism, has been calculated.

TABLE 5 Tested substance: MID y/cc. Daunosaminildaunomycin 6.25 Dihydrodaunomycin 12.50 Dihydrodaunomycinone 20 ANTIPROTOZOAL ACTIVITY The antiprotozoal activity has been tested in vitro on T richomonas foetus and Entamoeba lzistolytica and the Inhibiting B05350 (ID which is the concentration of the substance capable of inhibiting the development 1n vitro of 50% of the tested organism, has been calculated.

TABLE 6 BEZl Trichomonas Eutamoeba 'lested substance foetus hzstolytzca Daunosaminildaunomycin 100 Dihydrodaunomyein 100 160 Dihydrodaunomycinone 100 40 The suspension of the strain Strepromyccs peucetius without limiting it.

Example 1 The suspension of the strain Srrepromyces peucetius var. carneus obtained from a lyophilized flask was used to inoculate 5-7 slants of a medium having the following composition:

Percent Saccharose 0.2 Bipotassium phosphate 0.2 Sodium nitrate 0.2 Magnesium sulphate heptahydrate 0.2 Dry yeast 0.5

Tap water to 100.

Percent Bacto-peptone 0.5 Dry yeast 0.3 Calcium nitrate 0.05

Tap water to 100.

The pH of the medium, uncorrected, was about 7. The flasks were sterilized in autoclave at C. for 20 minutes. The inoculation of each flask consisted in a portion of the surface coat of the mycelium, corresponding to about A of the surface of the slant, homogenized with 2 cc. of distilled water. The flasks were incubated at 28 C.

on a rotary shaker at 240 r.p.m. with an eccentricity of 35 mm. After 48 hours of incubation these flasks were used to inoculate the flasks of the production phase, each containing 40 cc. of the medium B, having the following composition:

Percent Glucose 6 Dry yeast 2.5 Sodium chloride 0.2 Calcium carbonate 0.2 Dipotassium hydrogen phosphate 0.1 Anhydrous magnesium sulphate 0.01 Iron sulphate heptahydrate 0.001 Zinc sulphate heptahydrate 0.001 Copper chloride 0.001 Ethylendiaminotetraacetic acid neutralized with soda 0.012

Tap water to 100.

The pH of the medium, uncorrected, was 7. It was sterilized in an autoclave at 110 C. for 20 minutes. (Glucose was sterilized separately at 110 C. for 20 minutes.) The inoculation of the production flasks consisted in 2 cc. of the cultures of the vegetative phase. The fermentation conditions were the same used for the vegetative cultures. After hours of incubation, the culture contained 114 ,ug./cc. of total pigments (calculated as dihydrodaunomycin on the basis of the adsorption at 496 m of the ethanol extract of an aliquot part of the culture) which include 21 ,ug./cc. of dihydrodaunomycin, 4 ,ug./cc. of daunosaminildaunomycin and 10 g/cc. of dihydrodaunomycinone.

Example 2 Operation is as in Example 1 with the diflerence that for the vegetative and productive phases, the media L and D respectively were employed. These media have the following composition:

Vegetative medium L: Percent Glucose 1 Broth vegetal extract 1.5 Casein enzymatic hydrolyizate 0.3

Tap water to 100.

The pH was not corrected. The sterilization was carried out at 120 C. for 20 minutes.

Productive medium D. Percent Glucose 6 Distillers solubles 2.5 Sodium chloride 0.2 Calcium carbonate 0.2 Dipotassium hydrogen phosphate 0.1 Anhydrous magnesium sulphate 0.01 Iron sulphate heptahydrate 0.001 Zinc sulphate heptahydrate 0.001

Tap water to 100.

The pH was corrected to 7 with diluted NaOH. Sterilization was carried out at 110 C. for 20 minutes. The maximum concentration of dihydrodaunomycin, daunosaminildaunomycin and dihydrodaunomycinone was reached on the 6th day of fermentation and it was 66 g/ cc. of total pigments consisting of 21 gjcc. of dihydrodaunomycin, 3,ug./ cc. of daunosaminildaunomycin and 8 ,ug/cc. of dihydrodaunomycinone.

Example 3 Operating as in Example 1 with the difference that for the productive phase the medium employed has the following composition:

Percent Glucose 7 Chick peas meal 6.6 Calcium carbonate 0.2 Sodium chloride 0.2 Dipotassium hydrogen phosphate 0.1

Percent Magnesium sulphate heptahydrate 0.02 Iron sulphate heptahydrate 0.001 Zinc sulphate heptahydrate 0.001 Tap water to 100.

Sterilization was carried out at 120 C. for minutes. On the fifth day, we reached the concentration of 72 ,ug./CC. of total pigments, comprising 22 ,ug./cc. of dihydrodaunomycin, 4 ,ug./cc. of daunosaminildaunomycin and 9 ,ug./ cc. of dihydrodaunomycinone.

Example 4 (A culture of Streptomyces peucetius var. carneus on the solid medium described in Example 1 had been used to inoculate 500 cc of the liquid medium for the Vegetative phase described in Example 1, contained in a 2000 cc. flask of embossed Pyrex glass. The mycelium of the slant had been homogenized before inoculation. The flask had been incubated at 28 C. on a rotary shaker at 120 r.p.m., with an eccentricity of 35 mm. for 48 hours. 500 cc. of the vegetative culture thus obtained were used to inoculate, in an 80 liters stainless steel fermenter, 50 liters of a medium consisting of:

Percent Peptone 0.5 Dry yeast 0.3 Calcium nitrate tetrahydrate 0.05

Tap Water to 100.

The fermenter was shaken with turbine disc propellers at 250 rpm. and aerated with about 0.7 liter of air per liter of medium per minute at 27 C. After 48 hours of incubation, 25 liters of the mycelium suspension obtained were used to inoculate 500 liters of a fermentation medium in an 800 liters container. The medium had the following composition:

Tap water to 100.

The medium was previously sterilized at 120 C. and, after inoculation, it was shaken with two turbine disc propellers at 220 r.p.m., aerated with 0.7 liter of air per liter of medium per minute at 27 C. After 130-140 hours of incubation a potency of 69 ig/cc. of total pigments was reached.

50 liters of a filtered fermentation broth, containing 69 ,ug./CC. of total pigments, corresponding to a total of 3.45 g., were adjusted to pH 2 with 6 N hydrochloric acid and 5 liters of chloroform were added. After 20 minutes of shaking and after separation of the phases, the chloroform extract was collected and evaporated to dryness. The residue, washed with petroleum ether, consisted of 874- mg. of a red-brown product which, after two crystallizations from glacial acetic acid, gave 240 mg. of dihydro daunomycinone having melting point at 230- 232 C. [u] -=+75 C. (c.=0.05 in dioxane).

A mixture of 20 cc. of salicylic aldehyde and 200 cc. of methyl alcohol was added to the acid aqueous phase, corresponding to 52 g/cc. of total pigments; pH was adjusted to 7.5 with 32% ammonium hydrate. After minutes of shaking, pH was adjusted to 8.6. The mixture 'was then extracted with 10 liters of chloroform and shaken for an hour. After separating the organic phase, the extraction was twice repeated with a total amount of 8 liters of the solvent. The extracts collected were concentrated under reduced pressure to about 1.5 liters. They contained 2.58 g. of total pigments determined spectrophotometrically. The extracted solution was then evaporated and the residue was taken up with a small amount of methylene chloride and adsorbed over a column of 75 g. of silicic acid. The column was then eluted with the solvent system methylene chloride/ petroleum ether/methanol (:50zl0). Two colored bands were formed. From the more slowly descending colored band 1.65 g. of the amorphous salicyden derivative of dihydrodaunomycin were obtained. This product, crystallized from methanol, gave 1.23 g. of crystals melting at 165-168 C.; found: C, 64.24; H, 5.61; N, 2.13; OCH 4.86. Calculated per C H O N: C, 64.44; H, 5.58; N, 2.21; OCH 4.90.

In order to obtain the dihydrodaunomycin hydrochloride, 1 g. of salicylinden derivative was dissolved in 50 cc. of chloroform and extracted three times each with 15 cc. of 0.1 N HCl. The color transferred completely in the aqueous phase, which was made alkaline to a pH 8.6 with 0.1 N NaOH and extracted with chloroform till the extracts were only slightly colored. The chloro form extracts collected and dried over anhydrous sodium sulphate were concentrated under vacuum to about 10 cc. Added were 2.9 cc. of 0.6 N HCl in methanol and ten volumes of anhydrous ethyl ether. The precipitate obtained was crystallized from methanol-dioxane. Yield 0.52 g. of dihydrodaunomycin hydrochloride melting at 204-206 C. (with decomposition) [a] =+188 (c.=0.13 in methanol).

By elution of the first colored band of the above-cited column of silicic acid, 800 mg. of an amorphous product consisting of a mixture of pigments were obtained. The product was dissolved in 40 cc. of chloroform and extracted three times each with 10 cc. of 0.1 N HCl. In this way all the pigments were transferred into aqueous phase which was made alkaline to pH 8.6 with 0.1 N NaOH and extracted with chloroform. The three extractions were carried out for total amount of 100 cc. of solvent. The extracts collected together and dried over anhydrous sodium sulphate were concentrated under vacuum to 5 cc., then 1.5 cc. of 0.6 N HCl in methanol and ten volumes of anhydrous ethyl ether were added. 400 mg. of a precipitate were obtained which consisted of a crude hydrochloride which was purified over a column of 100 g. of cellulose powder (Whatrnan CF 11) buffered with a M/15 phosphate buifer at a .pH of 5.4 eluting with butyl alcohol saturated with M/15 phosphate butter at pH 5.4. Fractions of 25 cc. each were collected. From the fractions 21-50, 85 mg. of a product which consisted partially of daunosarninildaunomycin were obtained. The fractions 51-78 contained pure daunosaminildaunomycin were obtained. The fractions 51-78 contained pure daunosaminildaunomycin. To these fractions added together, were added 350 cc. of ethyl ether and 300 cc. of petroleum ether. It was then extracted three times with water, employing an amount of 450 cc. water. The aqueous phase, at pH 5.4, which now contained all the pigments, was extracted with chloroform and the extract was discarded. The aqueous phase was adjusted to a pH of 8.6 with 0.1 N NaOH. It was extracted with chloroform. Three extractions were carried out for a total amount of 600 cc. The chloroform extracts collected together were dried over anhydrous sodium to about 20 cc. To the concentrate 1.5 cc. of 0.6 N HCl in methanol and ten volumes of anhydrous ethyl ether were added. 200 mg. of amorphous daunosaminildaunomycin hydrochloride were obtained. The precipitate, recrystallized from met'hanol-isopropanol, gave mg. of crystals of daunosaminildaunornycin hydrochloride melting at 200 C. (with decomposition), [a] -=+142 (c.=0.1 in M OH).

We claim:

1. An antibiotic substance having the formula:

o on 1 I1 l O: OH

\I/ 00113 (i 0H --12,

wherein when R is OH E R is selected from the group consisting of the and of hydrogen,

'when R is =0, R is the daunosaminil radical and a second radical daunosaminil estcrifies one of the free hydroxy groups of the molecule,

and its salts with pharmaceutically acceptable non-toxic acids.

2. The compound of claim 1, which is dihydrodaunomycin.

3. The compound of claim 1, which is dihydrodaunomycin hydrochloride.

4. The compound of claim 1, which is daunosaminildaunomycin.

5. The comfpound of claim 1, which is daunosaminildannomycin hydrochloride.

6. The compound of claim 1, which is dihydrodaunomycmone.

References Cited UNITED STATES PATENTS 3,590,028 6/1971 Arcamone et a1. 260210 AB OTHER REFERENCES Iwamoto et al.: Tetrahedron Letter, No. 36, 1968, pp. 3891-3894.

LEWIS GOTTS, Primary Examiner J. R. BROWN, Assistant Examiner US. Cl. X.R.

195-80; 2602l0 R, 376, 393; 424l 

